bioRxiv preprint doi: https://doi.org/10.1101/174888; this version posted August 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Synthetic protein-sensing riboswitches Authors: Noa Katz1, Beate Kaufmann1, Roni Cohen1, Oz Solomon1,2, Orna Atar1, Zohar Yakhini2,3, Sarah Goldberg1, and Roee Amit1,4* Affiliation: 1Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa, Israel 32000. 2Department of Computer Science, Technion - Israel Institute of Technology, Haifa, Israel 32000. 3School of Computer Science, Interdisciplinary Center, Herzeliya, Israel. 4Russell Berrie Nanotechnology Institute, Technion - Israel Institute of Technology, Haifa 32000. *Correspondence to: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/174888; this version posted August 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract We study translational regulation by a 5’ UTR sequence encoding the binding site of an RNA-binding protein (RBP), using a reporter assay and SHAPE-seq, in bacteria. We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages MS2, PP7, GA, and Qβ, positioned in the 5’ UTR. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site. SHAPE-seq data for a representative construct exhibiting up-regulation indicate a partially- folded hairpin and a hypo-modified upstream flanking region, which we attribute to an intermediate structure that apparently blocks translation. RBP binding stabilizes the fully-folded hairpin state and thus facilitates translation. This indicates that the up-regulating constructs are RBP-sensing riboswitches. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state. Finally, the combination of two binding sites, positioned in the 5’ UTR and N-terminus of the same transcript can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behavior. This implies that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation. Keywords Riboswitch, RNA-binding protein, RBP-RNA interactions, phage coat proteins, MS2, PP7, translational repression, hairpin, translation stimulation. 2 bioRxiv preprint doi: https://doi.org/10.1101/174888; this version posted August 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. One of the main goals of synthetic biology is the construction of complex gene regulatory networks. The majority of engineered regulatory networks have been based on transcriptional regulation, with only a few examples based on post-transcriptional regulation (Win and Smolke, 2008; Green et al., 2014; Xie et al., 2011; Wroblewska et al., 2015). RNA-based regulatory components have many advantages. Several RNA components have been shown to be functional in multiple organisms (Harvey et al., 2002; Suess et al., 2003; Desai and Gallivan, 2004; Buxbaum et al., 2015). RNA can respond rapidly to stimuli, enabling a faster regulatory response than that possible at the transcriptional level (Hentze et al., 1987; St Johnston, 2005; Saito et al., 2010; Lewis et al., 2017). The response range of RNA components can be very wide (Green et al., 2014). RNA molecules form a variety of stable secondary and tertiary structures, which support diverse functions. Moreover, a single RNA molecule may contain multiple functional structures, which enables modularity. For example, distinct sequence domains within a molecule (Lewis et al., 2017; Khalil and Collins, 2010) may target different metabolites or nucleic acid molecules (Isaacs et al., 2006; Werstuck and Green, 1998). All of these characteristics make RNA an appealing target for engineered applications (Hutvágner and Zamore, 2002; Rinaudo et al., 2007; Delebecque et al., 2011; Xie et al., 2011; Chen and Silver, 2012; Ausländer et al., 2014; Green et al., 2014; Sachdeva et al., 2014; Pardee et al., 2016). Perhaps the most well-known class of RNA-based regulatory modules are ribsowitches (Werstuck and Green, 1998; Winkler and Breaker, 2005; Wittmann and Suess, 2012; Serganov and Nudler, 2013). Riboswitches are noncoding mRNA segments that regulate the expression of adjacent genes via structural change, effected by a ligand or metabolite. However, response to metabolites cannot be easily used as the basis of a regulatory network, as there is no convenient feedback or feed-forward mechanism for connection of additional network modules. Riboregulators (Buskirk et al., 2004; Bayer and Smolke, 2005; Green et al., 2014), namely riboswitches that sense a nucleic acid molecule, provide a transcription-based feedback mechanism, which can result in slow temporal dynamics (Green et al., 2014). Implementing network modules using RNA-binding proteins (RBPs) could enable multicomponent connectivity without compromising response time or circuit stability. Regulatory networks require both inhibitory and up-regulatory modules. The vast majority of known RBP regulatory mechanisms are inhibitory (Cerretti et al., 1988; Lim and 3 bioRxiv preprint doi: https://doi.org/10.1101/174888; this version posted August 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Peabody, 2002; Romaniuk et al., 1987; Brown et al., 1997; Sacerdot et al., 1998, Schlax et al., 2001). A notable exception is the phage RBP Com, whose binding was demonstrated to destabilize a sequestered ribosome binding site (RBS) of the Mu phage mom gene, thereby facilitating translation (Hattman et al., 1991; Wulczyn and Kahmann, 1991). Several studies have attempted to engineer activation modules utilizing RNA-RBP interactions, based on different mechanisms: recruiting the eIF4G1 eukaryotic translation initiation factor to specific RNA targets via fusion of the initiation factor to an RBP (Gregorio et al., 1999; Boutonnet et al., 2004), adopting a riboswitch-like approach (Ausländer et al., 2014), and utilizing an RNA-binding version of the TetR protein (Goldfless et al., 2012). However, despite these notable efforts, RBP- based translational stimulation is still difficult to design in most organisms. In recent work (Katz et al., 2017), we employed a synthetic biology and in vivo SHAPE- seq approach (Lucks et al., 2011; Spitale et al., 2013; Flynn et al., 2016) to study repression controlled by an RBP bound to a hairpin within the N-terminus of a reporter gene, in bacteria. Here, we focus on regulation by an RBP bound within the 5’ UTR of bacterial mRNA, following a design introduced by (Saito et al., 2010). Our findings indicate that structure-binding RBPs [coat proteins from the bacteriophages GA (Gott et al., 1991), MS2 (Peabody, 1993), PP7 (Lim and Peabody, 2002), and Qβ (Lim et al., 1996)] can generate a range of translational responses, from previously-observed down-regulation (Saito et al., 2010) to, surprisingly, up-regulation. The mechanism for downregulation is most likely steric hindrance of the initiating ribosome by the RBP-hairpin complex. For the 5’ UTR sequences that exhibit up-regulation, RBP binding seems to facilitate a transition from an RNA structure with a low translation rate, into another RNA structure with a higher translation rate. These two experimental features indicate that the up-regulatory elements constitute protein-sensing riboswitches. Our findings imply that RNA- RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation. Results RBP-binding can effect either up- or down-regulation We studied the regulatory effect generated by RNA binding phage coat proteins for GA (GCP), MS2 (MCP), PP7 (PCP), and Qβ (QCP) when co-expressed with a reporter construct 4 bioRxiv preprint doi: https://doi.org/10.1101/174888; this version posted August 10, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. containing one of eleven putative binding sites in the 5’ UTR [see Supp. Fig. 1 and (Johansson et al., 1998; Lim and Peabody, 2002)]. We positioned each of the 11 binding sites at three or four different locations upstream of the RBS, that varied from δ=-21 to δ=-31 nt measured relative to the ATG of the mCherry reporter gene (see Table S1). Altogether, we designed 44 reporter constructs (including non-hairpin controls), and co-transformed with all four RBPs, resulting in a total of 176 regulatory strains. RBP levels were induced by addition of N-butyryl-L-homoserine lactone (C4HSL), at 24 different concentrations. The normalized dose response functions for the 5’ UTR constructs are plotted as a heatmap in Fig. 1a in order of increasing response, with the highest-repression variants depicted at the bottom. There are striking differences between the dose-response behaviors observed for the 5’ UTR (Fig. 1a) and N-terminus configurations (Katz et al., 2017). First, the observed repression is significantly weaker for the 5' UTR hairpins, and at most amounts to about a factor of two reduction from basal levels.